WO2018220228A1

WO2018220228A1 - Method for producing aliphatic linear alpha-, omega- dinitriles from the corresponding dioximes using an aldoxime dehydratase

Info

Publication number: WO2018220228A1
Application number: PCT/EP2018/064620
Authority: WO
Inventors: Tobias Betke; Harald GRÖGER
Original assignee: Universität Bielefeld
Priority date: 2017-06-02
Filing date: 2018-06-04
Publication date: 2018-12-06
Also published as: DE102017112191A1

Abstract

The method relates to the production of dialdehyde dioxime precursors corresponding to medium chain linear α-, ω-dinitriles by means of an enzymatic conversion.

Description

PROCESS FOR THE PREPARATION OF ALIPHATIC LINEAR ALPHA, OMEGA DINITRILES FROM THE CORRESPONDING DIOXIMES USING AN ALDOXIMDEHYDRATASE

Description

The present invention relates to production processes of linear dinitriles.

Such compounds have great industrial importance, e.g. as precursors in the preparation of polyamides, polyurethanes or polyureas. Common methods, such as the addition of hydrogen cyanide (hydrocyanic acid, HCN) to butadiene to produce adiponitrile (adiponitrile, ADN), but often include harsh conditions or the use of highly toxic hydrogen cyanide, so that there is a constant need for alternative production processes of aliphatic linear α, ω-dinitriles, and this represents a task for the skilled person.

This object is achieved by a manufacturing method according to claim 1 of the present invention. Accordingly, a production method of aliphatic linear α, ω-dinitriles comprising> 5 and <12 carbon atoms is proposed, comprising the step

Reaction of the corresponding aliphatic, linear α, ω-dialdehyde dioximes with an aldoxime dehydratase. Surprisingly, it has been found that corresponding dinitriles can be prepared in a simple manner in good, sometimes even up to quantitative yields, while in particular the corresponding α, ω-Dialdehyddioxime be reacted with fewer carbon atoms only poorly or partially not at all to the corresponding dinitriles.

It has been found that for most applications within the present invention one or more of the following advantages can be achieved:

- The method is feasible at room temperature and does not require highly toxic and problematic cyanide or also highly toxic and problematic

Hydrogen cyanide

- The process is industrially applicable

The process proceeds under mild reaction conditions and has excellent product selectivity, with no by-products being typically observed. This advantage is therefore in contrast to previously common, today technically used production process for example adiponitrile (ADN), where usually the selectivity in the reaction and / or the decomposition of the product under the required reaction conditions is a limitation (see also: H J. Arpe, Industrial Organic Chemistry, 6th Edition, Wiley-VCH, Weinheim, 2007, 270-276.).

- The process is heavy-metal-free and is thus in contrast to the nowadays industrially used, developed by DuPont method of butadiene hydrocyanation, in which Ni (0) complexes with phosphine or phosphite ligands are used (for this DuPont method, see also: H.-J. Arpe, Industrial Organic Chemistry, 6th Edition, Wiley-VCH, Weinheim, 2007, 272-273.).

- The method allows in contrast to the above-mentioned method of Ni (0) - catalyzed butadiene hydrocyanation, which requires an organic solvent, a run in water as the reaction medium.

The term "aliphatic, linear α, ω-dinitriles" is understood in particular to mean compounds of the following structure: NC- [CH ₂ ] _n -CN with n> 3 to n <10 corresponding to> 5 and <12 carbon atoms of the dinitrile.

N> 4 to n <8, corresponding to a dinitrile with> 6 and <10, is particularly preferred

Carbon atoms, most preferably n> 4 to n <6, corresponding to a dinitrile having> 6 and <8 carbon atoms. The term "aliphatic linear α.ω-Dialdehyddioxime" are understood accordingly the following structures

HO-N = CH- [CH ₂ ] _n -CH = N-OH with n as above. Such compounds can be obtained in a simple manner, for example from the respective dialdehydes in the context of a condensation reaction with the hydroxylamine readily available in industrial quantities (as a synthesis unit used in ε-caprolactam production). Alternatively, instead of the dialdehydes, corresponding bis-acetals can also be used, and the dialdehydes required for oxime formation via condensation with hydroxylamine can be released in situ only by hydrolysis.

By "aldoxime dehydratase" is meant in particular enzymes and / or enzyme mutants which are capable of reacting oximes, ie compounds having the structure HO-N = CH-R, where R is any organic radical, to form nitriles Catalyze water elimination. Particularly preferred enzymes are aliphatic aldoxime dehydratases (eg the Pseudomonas chlororaphis B23 (OxdA), EC 4.99.1.5, according to the EC nomenclature of the "Nomenclature Committee of the International Union of Biochemistry and Molecular Biology"), phenylacetaldoxime dehydratases (eg the aldoxime dehydratase from Bacillus sp., OxB-1 (OxdB), EC 4.99.1.7) or Indole acetaldehyde dehydratases ("indoleacetaldoxime dehydratase"; EC 4.99.1.6) Particularly preferred are aldoxime dehydratases from Pseudomonas chlororaphis B23 (OxdA) and / or Bacillus sp .OxB-1 (OxdB). [0032] The reaction of the dialdehyde dioximes can be carried out according to a preferred embodiment with isolated enzyme ,

According to an alternative, but also preferred embodiment, the reaction is carried out with cells in which corresponding aldoxime dehydratases are overexpressed, in particular corresponding E. coli cells. Such cells are also called recombinant

"Whole Cell Catalysts".

The reaction can be carried out according to a preferred embodiment in water or aqueous solution containing the usual buffering and Hilfsreagentien. Preferred buffers here are e.g. Phosphate buffer or other buffer systems that can operate at pH> 6.5 to <7.5.

Preferably, during the reaction, the pH of> 6.5 to <7.5 is preferably> 6.9 to <7.1. According to an alternative but also preferred embodiment, the reaction takes place in a mixture of water or aqueous solution and at least one polar

Solvent, with a Ετ30 value of> 45.

"Ετ30 value" is understood to mean the polarity of a solvent, measured in accordance with C. Reichardt, Chem. Rev. 1994, 94, 2319-2358.

Preferred polar solvents are DMSO and short chain primary alcohols, especially methanol and ethanol. According to a preferred embodiment, the proportion of total polar

Solvents> 10% and <25% (in vol / vol of the total solvent), preferably> 15% and <

20%.

According to another alternative, but also preferred embodiment, the reaction takes place in a purely organic solvent system, wherein this also

Solvent mixtures of several organic solvents may mean.

The above-mentioned and the claimed components to be used according to the invention described in the exemplary embodiments are not subject to special conditions of size, shape, material selection and technical design, so that the selection criteria known in the field of application can be used without restriction.

Further details, features and advantages of the subject matter of the invention will become apparent from the subclaims and from the following description of the accompanying examples, which are purely illustrative and not restrictive.

1. General Procedure 1 for the Synthesis of Dialdehyde Dioximes (AAV 1)

n = 1, 2.4 n = 3

n = 1: 28% yield n = 2: 64% yield n = 3: 66% yield n = 4: 78% yield

Hydroxylamine hydrochloride (3.0 eq.) Was dissolved in H ₂ O at room temperature. The solution was degassed and overlaid with an argon atmosphere. The corresponding Bis (dimethyl) acetal or the corresponding dialdehyde was added and the resulting suspension was heated to 40 ° C. After clarifying the suspension to a solution, sodium carbonate (1.5 eq.) Was added after five minutes. This precipitated a colorless solid. The suspension was stored at 0 ° C. for 24 hours and then the solid was filtered off and washed with water. The solid could without further

Purification can be used. Eventual slight contamination of the solid was recrystallized from ethanol. The dioximes are almost exclusively in the Z, Z configuration. Malonoaldehyde dioxime starting from 1,1,3,3-tetramethoxypropane

The synthesis was carried out according to AVV 1. 1,1,3,3-Tetramethoxypropane (5.00 mL, 30.4 mmol) was added to a solution of hydroxylamine hydrochloride (6.34 g, 91.2 mmol) in 20 mL H ₂ 0. Heating to 40 ° C gave a yellow solution and sodium carbonate (4.83 g, 45.6 mmol) was added. Work up according to AVV 1 gave the product as a colorless solid. The E / Z ratio (concerning both oxime groups) was 7:93 (determined by ¹ H-NMR spectroscopy).

Yield: 744 mg, 28%. Succinaldehyde dioxime starting from 1,1,4,4-tetramethoxybutane

The synthesis was carried out according to AVV 1. 1,1,4,4-Tetramethoxybutane (2.65 mL, 15.0 mmol) was added to a solution of hydroxylamine hydrochloride (3.13 g, 45.0 mmol) in 10 mL H ₂ 0. Heating to 40 ° C gave a colorless solution and sodium carbonate (2.38 g, 22.5 mmol) was added. Work up according to AVV 1 gave the product as a colorless solid. The E / Z ratio (concerning both oxime groups) was 8:92 (determined by ¹ H NMR spectroscopy).

Yield: 1.12 g, 64%.

Glutaraldehyde dioxime from glutaraldehyde A 50 wt% solution of glutaraldehyde (2.00 mL, 11.2 mmol) was added to a solution of hydroxylamine hydrochloride (2.34 g, 33.6 mmol) and sodium carbonate (3.56 g,

33.6 mmol) in 50 mL H ₂ 0. After 30 minutes, the solution became a colorless suspension. Work up according to AVV 1 gave the product as a colorless solid. The EIC ratio (for both oxime groups) was 8:92 (determined by Ή-ΝΜΡν spectroscopy).

Yield: 960 mg, 66%. Adipaldehyde dioxime starting from 1,1,6,6-tetramethoxyhexane

The synthesis was carried out according to AVV 1. 1,1,6,6-Tetramethoxyhexane (824 μί, 4.00 mmol) was added to a solution of hydroxylamine hydrochloride (834 mg, 12.0 mmol) in 10 mL H ₂ 0. Heating to 40 ° C gave a colorless solution and sodium carbonate (636 mg, 6.00 mmol) was added. Work up according to AVV 1 gave the product as a colorless solid. The E / Z ratio (for both oxime groups) was 43:57 (as determined by NMR NMR spectroscopy). Alternatively, larger amounts of adipaldehyde dioxime can be synthesized from adipaldehyde, which is readily available according to a literature protocol (S. Lopez, F. Fernandez-Trillo, P. Midon, P. Castedo, C. Saa, J. Org. Chem. 2005, 70, 6346-6352).

Yield: 450 mg, 78%.

2. Expression of the Aldoxime Dehydratases Preculture: 10 mL LB medium in 100 mL Erlenmeyer flasks were mixed with an antibiotic concentration of 50 μg / mL kanamycin or 100 μg / mL carbenicillin and 34 μg / mL chloramphenicol. Subsequently, an E. coli clone was added and incubated for 24 hours at 37 ° C and 180 rpm.

Main culture: 100-450 mL (in 100-500 mL Erlenmeyer flasks) Auto-induction medium was inoculated with 1.0% by volume of the preculture followed by addition of the antibiotics up to a final concentration of 50 μg / mL kanamycin or 100 μg / mL carbenicillin and

34 μg / mL chloramphenicol. The culture was incubated for one hour at 37 ° C and 180 rpm. It was then incubated at 15 ° C (OxdA) or 30 ° C (OxdB) for 72 hours.

The cells were harvested by centrifugation (4000 g, 4 ° C, 15 min). The supernatant was removed and the cell pellet washed twice with 50 mM KPi buffer (pH = 7.0). After re-centrifugation of the pellet (4000 g, 4 ° C, 15 min), it was resuspended in 50 mM 50 mM KPi buffer (pH = 7.0), optionally overlaid with argon and stored at 4 ° C until use.

Table 1: Plasmids used for the transformation of E. coli BL21-CodonPlus (DE3) -RIL.

No vector / plasmid origin aldoxime resistance

organism dehydratase

pET28_OxdA (C) Pseudomonas oxdA kanamycin

chlororaphis

B23

pUC18_OxdB Bacillus sp. OxdB Carbenicillin

OXB-1

3. Preparation of the dinitriles

3.1. General Working Regulation 2 (based on adipaldehyde dioxime) (AAV 2)

Whole-cell catalyst

30 ° C, argon, if necessary.

20% by volume DMSO

75-375 mM> 99% conversion,

55-80% yield In a sealable reaction vessel (100 ml), 100 ml of a suspension of recombinant whole-cell catalysts (50 mM KPi buffer, pH = 7.0) in which the

Aldoxime Dehydratase OxdA (from Pseudomonas chlororaphis, see Y. Miao, R. Metzner, Y.

Asano, ChemBioChem, 2017, 18, 451-454) or OxdB (from Bacillus sp., OxB-1, see Y. Kato, Y. Asano, Protein Expression and Purification, 2003, 28, 131-139) , When using DMSO as cosolvent 80 mL cell suspension and 20 mL DMSO were submitted. Subsequently, E, Z-adipaldehyde dioxime (final concentration of 75 mM-375 mM) was added to this suspension. The reaction mixture was overlaid with argon and shaken under this inert gas atmosphere at 30 ° C at 180 rpm. The conversion was checked by gas chromatography.

The reaction mixture was extracted three times with MTBE (1: 1, v / v) at complete conversion. When DMSO was used, the combined extracts were washed with sat. NaCl solution (1: 1, v / v). After drying over MgSO ₄ and removal of the solvent in vacuo, the product was obtained as a pale yellow liquid with a purity of> 99.

3.2. Implementation in 75mM scale (-10g / L)

After the above procedure AAV 2 E, Z-adipaldehyde oxime (1.08 g, 7.50 mmol) was suspended in the cell suspension and shaken under inert gas atmosphere at 30 ° C.

Workup provided the product as a light yellow liquid.

Input aldoxime dehydratase / cell mass Reaction time Yield [%] if necessary Add solvent [mgßww] ³ [h]

1 OxdA / 20% DMSO 578 96 75 (608 mg)

2 OxdA 1156 64 59 (480 mg)

3 OxdB / 20% DMSO 507 18 55 (446 mg) 4 OxdB 507 15 70 (570 mg) a) BWW = bio wet mass

3.3. Biotransformations in the 50 g / L scale Following the above procedure AAV 2, E, Z-adipaldehyde oxime (5.00 g, 34.7 mmol) was suspended in the cell suspension and shaken under inert gas atmosphere at 30 ° C.

Workup provided the product as a light yellow liquid.

Entry aldoxime dehydratase / cell mass reaction time yield [%] if necessary, solvent addition [mgßww] ³ [h]

ΪOxdB / 20% DMSO \ 5QQ 87 67 (2.47 g)

2 OxdB 1500 22 80 (2.91 g) a) BWW = organic moisture

3.4. Synthesis experiments of malononitrile and succinonitrile in 10-75 mM scale

10-75 mM up to 20% by volume DMSO _no conversion to the dinitrile

Malonaldehyde dioxime and succinaldehyde dioxime were analyzed on an analytical scale (1.0 mL reaction volume, 10 mM substrate concentration) in a suspension of whole cell recombinant catalysts (50 mM KPi buffer, pH = 7.0) containing the aldoxime dehydratase OxdA (from Pseudomonas chlororaphis) or OxdB (from Bacillus sp OxB-1) was overexpressed. The DMSO content here was 2.5-20% by volume. After 24 hours, the reaction mixture was extracted with 2-methyltetrahydrofuran and analyzed. Loud

Gas chromatography could be detected for Malonaldehyddioxim no sales and For Succinaldehydoxim also no conversion to dinitrile, only a small amount of Mononitrilspezies was formed.

On a preparative scale (100 mL reaction volume, 75 mM substrate concentration), no conversion to the dinitrile was also observed for succinonitrile after 24 hours.

The individual combinations of the components and the features of the already mentioned embodiments are exemplary; the exchange and substitution of these teachings with other teachings contained in this document with the references cited are also expressly contemplated. The person skilled in the art recognizes that variations,

Modifications and other embodiments described herein may also occur without departing from the spirit and scope of the invention. Accordingly, the above description is illustrative and not restrictive. The word "comprising" used in the claims does not exclude other ingredients or steps The indefinite article "a" does not exclude the meaning of a plural. The mere fact that certain measures are recited in mutually different claims does not make it clear that a combination of these measures can not be used to the advantage. The scope of the invention is defined in the following claims and the associated equivalents.

The invention underlying this patent application was developed in a project which was funded by the BMBF under grant number D-2110-0301-0017-2100.

Claims

P a n t a n s p r e c h e

A process for preparing aliphatic linear α, ω-dinitriles comprising> 5 and <12 carbon atoms, comprising the step:

Reaction of the corresponding aliphatic linear α, ω-dialdehyde dioximes with an aldoxime dehydratase.

The process of claim 1 wherein the aliphatic linear α, ω-dinitriles comprise> 6 and <10 carbon atoms

A process according to claim 1 or 2, wherein during the reaction the pH is from> 6.5 to <7.5

Method according to one of claims 1 to 3, wherein the aldoxime dehydratase an aldoxime dehydratase from Pseudomonas chlororaphis B23 (OxdA) and / or Bacillus sp. OxB-1 (OxdB)

Method according to one of claims 1 to 4, wherein the reaction is carried out in a mixture of water or aqueous solution and at least one polar solvent, with a Ετ30 value of> 45.